Beloxepin and analogs for the treatment of pain

ABSTRACT

This present disclosure provides methods of treating pain with beloxepin and/or beloxepin analogs.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/941,371,filed Nov. 8, 2010, which is a continuation of application Ser. No.12/750,558, filed Mar. 30, 2010, which is a continuation of applicationSer. No. 12/388,982, filed Feb. 19, 2009, which claims the benefit under35 U.S.C. §119(e) of provisional application Ser. No. 61/029,916, filedFeb. 19, 2008, and provisional application Ser. No. 61/050,921, filedMay 6, 2008, the contents of all of which are incorporated herein byreference.

2. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

None.

3. PARTIES TO A JOINT RESEARCH AGREEMENT

None.

4. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

None.

5. BACKGROUND

Acute and chronic pain of both nociceptive and non-nociceptive originare disabling conditions that affect significant numbers of individuals.Pain is frequently characterized by increased sensitivity to normallynon-noxious stimuli (allodynia) and/or painful stimuli (hyperalgesia).Although antidepressants such as norepinephrine and serotonin (5HT)reuptake inhibitors have been used as a first-line therapy for treatingcertain types of pain, for example, pain associated with diabeticneuropathy, postherpetic neuralgia, fibromyalgia, irritable bowelsyndrome and interstitial cystitis, none of these therapies has provento be universally effective. Despite the number of therapies available,significant numbers of individuals still suffer debilitating pain on adaily basis. Accordingly, there is a need in the art for additionalcompounds and regimens useful for treating pain, whether acute orchronic, or due to nociceptive or non-nociceptive origin.

6. SUMMARY

Beloxepin, also known as “Org-4428” and“cis-1,2,3,4,4a,13b-hexahydro-2,10-dimethyldiben-[2,3:6,7]oxepino[4,5c]pyridine-4a-ol],” is a tetracyclic compound that underwentclinical evaluation as a potential antidepressant in the late 1990s.According to published reports, beloxepin is a highly specific inhibitorof noradrenaline reuptake in synaptosomes from rat and primate brain inin vitro assays, having greater than 100-fold less affinity for othermonoamine carriers (i.e., serotonin and dopamine transporters), and noor very weak affinity for noradrenergic, histaminergic and cholinergicreceptors (Sperling & Demling, 1997, Drugs of Today 33(2):95-102). It isalso reported to have modest affinity for the 5HT_(2C) receptor(Claghorn & Lesem, 1996, Progress Drug Res 46:243-262).

In preclinical studies with animal models of depression, beloxepin wasnoted to exhibit antidepressant properties by offsetting acquiredimmobility behavior, reserpine-induced hypothermia, and conditionedavoidance behavior. In these tests, beloxepin did not cause sedation,motor impairment or other untoward side effects. Its profile onEEG-defined sleep/wake behavior is compatible with that of a nonsedativeantidepressant with sleep-improving properties (Sperling & Demling,1997, supra). Results of sleep studies in human volunteers have shownthat beloxepin (25-400 mg) dose-dependently prolonged REM latency, bothacutely and sub-chronically, and decreased total duration of nocturnalREM sleep as recorded by EEG (Van Bemmel et al., 1999,Neuropsychobiology 40(2):107-114). No sedation or other side effectswere observed. Based on these studies, it was concluded that beloxepinmay reduce sleep continuity in depressed patients and may improve thedepth of sleep.

In a single-dose safety study, beloxepin displayed linear kinetics overa broad range, with a dose-independent t_(max) of one to four hours andt_(1/2) of 11 to 15 hr following doses of 10 to 500 mg. Steady-statepharmacokinetic parameters obtained in healthy normal subjects, whoparticipated in a multiple rising-dose safety and tolerance study,showed that at doses of 50 to 800 mg, t_(max) was 1.17 hr and t_(1/2)varied from 12 to 14 hr. No important adverse effects were observed inhealthy volunteers who received up to 800 mg/day of beloxepin. In aphase IIA study in patients hospitalized for depression, ⅔ of patientshad a moderate to good response, based on HAMD score reduction (Claghorn& Lesem, 1996, supra).

In subsequent clinical trials, beloxepin exhibited insufficient efficacyfor the treatment of major depression. Consequently further developmentof beloxepin was stopped (Paanakker et al., 1998, J. Pharm. Biomed.Anal. 16(6):981-989).

Affinity testing with over 125 receptors, channels and transportersindicates that beloxepin binds with only modest affinity to the NET(K_(i)=700 nM) and has only marginal affinity for the serotonintransporter (27% inhibition of binding at 10 μM in a competition assay)and dopamine transporter (16% inhibition of binding at 10 μM in acompetition assay). In a functional assay, beloxepin exhibited weakinhibition of norepinepherine reuptake (IC₅₀=130 nM).

Historically, antidepressants including those that inhibit reuptake ofNE (NRIs) and/or 5HT (SRIs) have been used as a first-line therapy fortreating both acute and chronic pain that is either nociceptive ornon-nociceptive in origin, for example, neuropathy, post-herpeticneuralgia (PHN), pain associated with fibromyalgia, pain associated withirritable bowel syndrome and interstitial cystitis (Sindrup and Jensen,1999, Pain 83(3):389-400; Collins et al., 2000, J. Pain & SymptomManagement 20(6):449-458; Crowell et al., 2004, Current Opin. Invest.Drugs 5(7):736-742). A recent study systematically evaluated therelative activity at the NE and/or 5HT transporter required for maximalefficacy in rodent models of pain (Leventhal et al., 2007, J. Pharmacol.Exper. Ther. 320(3):1178-1185). The effects observed replicate thoseobserved clinically for treating neuropathic pain conditions. Namely,compounds with greater affinity for the NE transporter are moreeffective at treating pain, and compounds with greater affinity for the5HT transporters have limited efficacy (see, e.g., Max et al., 1992; N.Engl. J. Med. 326(19):1250-1256; Collins et al., 2000, supra). Indeed,in a double-blind, placebo-controlled head-to-head study comparing thetetracyclic NRI maprotiline and the SRI paroxetine, reduction in painintensity was significantly greater for study completers randomized tomaprotiline (45%) as compared to paroxetine (26%) or placebo (27%)(Atkinson et al., 1999, Pain 83(2):137-145).

Given its weak affinity for the NET and its weak, albeit selective,inhibition of NE reuptake, beloxepin would not be expected to beeffective in treating pain. Surprisingly, the present inventors havediscovered that not only is beloxepin extremely effective in rodentmodels of various different pain syndromes, its antiallodynic activityis superior to that of known NRI compounds (e.g., reboxetine), dualNRI/SRI compounds (e.g., duloxetine) and tricyclic antidepressants(e.g., amitriptyline) currently used to treat pain when dosed at thesame concentrations via IP administration.

Indeed, the magnitude of tactile allodynia observed for beloxepin in theL5 SNL rodent model of pain at 30 min post treatment is amongst thehighest observed by the inventors in this model for drugs administeredIP. Also see FIG. 11 and Example 10, presenting a comparison of theantiallodynic effects observed upon administration beloxepin,duloxetine, and esreboxitine using the rat L5 SNL model system.

As demonstrated in FIG. 3, beloxepin produced an observed mean thresholdof approximately 15 g—nearly 5 times greater—under the same experimentalconditions than reboxetine. With reference to FIG. 2, beloxepin produceda tactile antiallodynic effect that was 852% greater than that observedwith vehicle-treated controls, and nearly 100% of that observed withsham-operated animals.

Beloxepin also exhibited extremely robust activity in rodent models ofacute nociceptive pain (FIGS. 6A and 6B), inflammatory pain (FIG. 7 andFIG. 9), neuropathic pain (FIG. 10 and Example 12), post-operativeincisional pain (FIG. 12, FIG. 13, FIG. 14, and Example 13), andvisceral pain (FIG. 8). For example, with reference to FIGS. 6A and 6B,beloxepin exhibited anti-nociceptive activity almost equivalent to thatof 3 mg/kg morphine. With reference to FIG. 7, beloxepin exhibitednearly complete reversal of hyperalgesia in rats treated with Freund'sComplete Adjuvant (FCA), and with reference to FIG. 8, beloxepininhibited acetic acid-induced writhing in mice a dose-dependent fashion.

The chemical structure of beloxepin is illustrated below:

The OH and H substituents attached to the carbon atoms marked withasterisks are in the cis configuration with respect to one another.These carbon atoms are chiral. As a consequence, beloxepin is a racemicmixture of two cis enantiomers, a (+) enantiomer and a (−) enantiomer.The absolute configurations about the chiral carbons of the (+) and (−)enantiomers are unknown.

Analogs of beloxepin are known in the art. For example, analogs ofbeloxepin are described in U.S. Pat. No. 4,977,158, the disclosure ofwhich is incorporated herein by reference. These analogs are expected toexhibit anti-pain activities similar to beloxepin.

Accordingly in one aspect, the present disclosure provide a method oftreating pain in a mammal comprising administering to a mammal sufferingfrom pain, including a human, an amount of beloxepin and/or a beloxepinanalog effective to treat the pain.

The beloxepin or beloxepin analog can be administered as the compoundper se, or in the form of a composition. The beloxepin or beloxepinanalog can be included in the composition as the free base, or in theform of a salt. In some embodiments the beloxepin and/or beloxepinanalog is included in the composition in the form of a pharmaceuticallyacceptable salt.

The composition can be formulated for administration to animals inveterinary contexts, or for administration to humans, via virtually anyroute or mode of administration, including, but not limited to, oral,topical, ocular, buccal, systemic, nasal, injection, transdermal,rectal, vaginal, inhalation or insufflation. In some embodiments, thecomposition is formulated for oral administration, for example, tohumans.

The methods can be used to treat numerous different types of painsyndromes, including acute or chronic pain that is either nociceptive(for example somatic or visceral) or non-nociceptive (for exampleneuropathic or sympathetic) in origin. In some embodiments, the pain isnociceptive pain including, but not limited to, surgical pain,inflammatory pain such as that associated with inflammatory bowelsyndrome (“IBS”) or rheumatoid arthritis, pain associated with cancer,and pain associated with osteoarthritis. In some embodiments, the painis non-nociceptive pain including, but not limited to, neuropathic painsuch as post-herpetic neuralgia (“PHN”), trigeminal neuralgia, focalperipheral nerve injury, anesthesia clolorosa, central pain (forexample, post-stroke pain, pain due to spinal cord injury or painassociated with multiple sclerosis), and peripheral neuropathy (forexample, diabetic neuropathy, inherited neuropathy or other acquiredneuropathies).

The beloxepin and/or beloxepin analog can be administered alone, or itcan be administered in combination with, or adjunctively to, one or moreother drugs useful for treating pain and/or other indications. Specificnon-limiting examples of drugs that can be used in combination with, oradjunctively to, the beloxepin and/or beloxepin analogs in a paintreatment or management regimen are provided in a later section. In onespecific embodiment, beloxepin is administered in combination with, oradjunctively to, one or more beloxepin analogs.

7. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a graph demonstrating the antiallodynic effect ofbeloxepin (30 mg/kg IP) in L5 SNL rats 14 days post surgery;

FIG. 2 provides a graph demonstrating the antiallodynic effect ofbeloxepin (3, 10 and 30 mg/kg IP) in L5 SNL rats 16 days post surgery;

FIG. 3 provides a graph illustrating the superior antiallodynic effectof beloxepin (30 mg/kg IP) as compared to reboxetine, a selectivenorepinephrine reuptake inhibitor (30 mg/kg IP), in L5 SNL rats;

FIG. 4 provides a graph demonstrating the antiallodynic effect of orallyadministered beloxepin (60 mg/kg PO) in L5 SNL rats 8 days post surgery;

FIG. 5 provides a graph comparing the antiallodynic effects produced bybeloxepin, duloxetine, amitriptyline, and reboxetine (each at aconcentration of 30 mg/kg IP) in L5 SNL rats;

FIGS. 6A and 6B provide graphs demonstrating the robust anti-nociceptiveactivity of beloxepin in a rodent model of acute nociception;

FIG. 7 provides a graph illustrating the robust antihyperalgesiaactivity of beloxepin in an animal model of inflammatory pain (ratstreated with Freund's Complete Adjuvent);

FIG. 8 provides a graph illustrating the robust activity of beloxepin ina rodent model of visceral pain (mice treated with acetic acid);

FIG. 9 provides a graph comparing the mechanical antihyperalgesiceffects of (30 mg/Kg IP) (±)-beloxepin and a reconstituted equimolar(racemic) mixture (30 mg/Kg IP) of (+)-beloxepin and (−)-beloxepin, inFCA-treated rats, 24 hours after FCA injection;

FIG. 10 provides a graph demonstrating the antiallodynic effect oforally administered beloxepin (60 mg/kg PO) in L5 SNL rats 7 days postsurgery;

FIG. 11 provides a graph comparing the antiallodynic effects ofbeloxepin, duloxetine, and esreboxetine (each compound dosed at 30 mg/kgIP) in L5 SNL rats;

FIG. 12 provides a graph demonstrating the antiallodynic effect ofbeloxepin (30 mg/kg IP) in the rat hindpaw incisional model 24 hourspost surgery;

FIG. 13 provides a graph demonstrating the antiallodynic effect oforally-administered beloxepin (60 mg/kg IP) in the rat hindpawincisional model 24 hours post surgery; and

FIG. 14 provides a graph demonstrating the antiallodynic effect ofintravenously-administered beloxepin (3 mg/kg IV) in the rat hindpawincisional model 24 hours post surgery.

FIG. 15 provides a graph illustrating the inhibition of CYP2D6(dextromethorphan O-demethylation) by beloxepin and quinidine.

8. DETAILED DESCRIPTION

The present disclosure concerns the use of beloxepin and/or its analogsto treat pain. The disclosure is based, in part, on the surprisingdiscovery that beloxepin, which is a weak selective inhibitor of NEreuptake, nonetheless produces significant and robust activity across abroad spectrum of rodent models of various types of pain syndromes,including rodent models of acute nociceptive pain, inflammatory pain,visceral pain and neuropathic pain. As discussed in the Summary,inhibition of NE reuptake correlates with efficacy in the treatment ofpain (see, Max et al., 1992, supra; Collins et al., 2000, supra;Atkinson et al., 1999, supra; Levental et al., 2007, supra). Based onits weak activity at the NET, beloxepin would not be expected to beuseful in treating pain. Yet, it produces robust activity in numerousanimal models of pain, and in the case of tactile anitallodynia,activity of magnitude greater than that observed with numerous compoundsknown to be effective in treating pain.

8.1 Beloxepin Compounds And Compositions

Beloxepin, also known as “Org-4428” and“cis-1,2,3,4,4a,13b-hexahydro-2,10-dimethyldiben-[2,3:6,7]oxepino[4,5c]pyridine-4a-ol],” is illustrated below:

The OH and H substituents attached to the carbon atoms marked withasterisks are in the cis configuration with respect to one another.Since these carbons are chiral, this cis geometric isomer is a racemicmixture of two enantiomers, a (+) enantiomer and a (−) enantiomer. Theabsolute configurations about the chiral carbons of these (+) and (−)enantiomers are not presently known.

Analogs of beloxepin have been reported in the art. For example, U.S.Pat. No. 4,977,158, the disclosure of which is incorporated herein byreference, discloses beloxepin analogs according to structural formula(I):

wherein:

-   -   n is 0 or 1;    -   X is O or S;    -   R¹ represents one or two identical or different substituents        selected from H, OH, halogen, C₁-C₄ alkyl and C₁-C₄ alkoxy;    -   R² represents one or two identical or different substituents        selected from H, OH, halogen, C₁-C₄ alkyl and C₁-C₄ alkoxy;    -   R³ and R⁴ are two substituents which are in the cis        configuration, where R³ is OH and R⁴ is H; and    -   R⁵ is H or C₁-C₄ alkyl.

These analogs are expected to have biological and pharmacologicalproperties similar to beloxepin, and are therefore also expected to beeffective in treating and managing various pain syndromes as describedherein. Beloxepin analogs according to structural formula (I) arereferred to herein as “beloxepin analogs,” or other grammaticalequivalents. Thus, the beloxepin analogs can be used in the variouscompositions and methods described herein and the various illustrativeembodiments described for beloxepin apply also to the beloxepin analogsas if such embodiments were specifically described.

Beloxepin and/or its analogs can be used in the various methodsdescribed herein as the compound per se, or can be included in acomposition formulated for, among other things, a specific mode ofadministration. The beloxepin or beloxepin analog can be present in thecomposition as the free base, or in the form of a salt, for example, anacid additional salt. In some embodiments, such salts arepharmaceutically acceptable salts.

Generally, “pharmaceutically acceptable salts” are those salts thatretain substantially one or more of the desired pharmacologicalactivities of the parent compound and which are suitable foradministration to humans. Pharmaceutically acceptable salts include, butare not limited to, acid addition salts formed with inorganic or organicacids. Inorganic acids suitable for forming pharmaceutically acceptableacid addition salts include, by way of example and not limitation,hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydriodic,etc.), sulfuric acid, nitric acid, phosphoric acid and the like. Organicacids suitable for forming pharmaceutically acceptable acid additionsalts include, by way of example and not limitation, acetic acid,trifluoroacetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl) benzic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.),arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-tuluenesulfonic acid,camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like.

8.2 Methods of Synthesis

Beloxepin and beloxepin analogs can be synthesized or prepared usingmethods described in the literature, for example, as described in U.S.Pat. No. 4,977,158, the disclosure of which is incorporated herein byreference. A specific method for synthesizing beloxepin that can beroutinely adapted to synthesize beloxepin analogs, the details of whichare discussed in the Examples section, is illustrated in Scheme 1,below:

8.3 Uses of Beloxepin and Its Analogs

Pain is generally understood to refer to the perception or condition ofunpleasant sensory or emotional experience, which may or may not beassociated with actual damage to tissues. It is generally understood toinclude two broad categories: acute and chronic (see, e.g., Analgesics,Buschmann et al., Wiley-VCH, Verlag GMbH & Co. KgaA, Weinheim, 2002;Jain, 2000, Emerging Drugs 5(2):241-257) that is either of nociceptiveorigin (for example somatic or visceral) or non-nociceptive origin (forexample neuropathic or sympathetic). Acute pain generally includesnociceptive pain arising from strains/sprains, burns, myocardialinfarction, acute pancreatitis, surgery, trauma and cancer. Chronic paingenerally includes nociceptive pain, including, but not limited to,inflammatory pain such as that associated with IBS or rheumatoidarthritis, pain associated with cancer and pain associated withosteoarthritis; and non-nociceptive pain, including, but not limited to,neuropathic pain such as post-herpetic neuralgia, trigeminal neuralgia,focal peripheral nerve injury, anesthesia clolorosa, central pain (forexample, post-stroke pain, pain due to spinal cord injury or painassociated with multiple sclerosis), and peripheral neuropathy (forexample, diabetic neuropathy, inherited neuropathy or other acquiredneuropathies).

Data presented in the Examples section confirm that beloxepin issurprisingly effective at treating pain in rodent models of neuropathic,acute nociceptive, inflammatory and visceral pain. Based upon thisanimal data, it is expected that beloxepin and beloxepin analogs will beuseful in treating various different pain syndromes including, but notlimited to, acute pain of nociceptive origin, such as, for example,surgical pain, chronic pain of nociceptive origin, such as, for example,inflammatory pain or cancer pain, and chronic pain of non-nociceptiveorigin, such as, for example, neuropathic pain.

In general, a “therapeutically effective” amount of a compound orcomposition is an amount that eradicates or ameliorates the underlyingdisease or indication being treated and/or that eradicates orameliorates one or more of the symptoms associated with the underlyingdisorder such that the patient reports an improvement in feeling orcondition, not withstanding that the patient may still be afflicted withthe underlying disease or indication. Therapeutic benefits also includeshalting or slowing the progression of the disease or indication,regardless of whether improvement is realized.

In the context of pain, a therapeutically effective amount is an amountof compound or composition that eradicates or ameliorates the pain orthe symptoms thereof, including, but not limited to, shootingsensations, burning sensations, electrical sensations, aching,discomfort, soreness, tightness, stiffness, sleeplessness, numbness, andweakness.

The therapy can be applied following the onset of pain and/or one ormore of its symptoms, or prophylactically to avoid or delay its onset.

8.4 Combination Therapies

Beloxepin and/or its analogs can be used alone, or in combination with,or adjunctively to, other therapeutic agents to treat pain.

Accordingly, beloxepin and/or its analogs can be combined with otheranalgesics, including but not limited to, cannabinoids and opioids. Anumber of cannabinoids are available that may be suitable for use incombination therapy, including, but not limited to, a cannabinoid thatis selected from a Δ⁹-tetrahydrocannabinol and cannabidiol, and mixturesthereof.

Alternatively, beloxepin and/or its analogs may be used in combinationwith at least one opioid. A wide variety of opioids are available thatmay be suitable for use in combination therapy to treat pain. As such,the combination therapy may involve an opioid that is selected from, butnot limited to, alfentanil, allylprodine, alphaprodine, anileridine,benzyl-morphine, bezitramide, buprenorphine, butorphanol, clonitazene,codeine, cyclazocine, desomorphine, dextromoramide, dezocine,diampromide, diamorphone, dihydrocodeine, dihydromorphine, dimenoxadol,dimepheptanol, dimethylthiambutene, dioaphetylbutyrate, dipipanone,eptazocine, ethoheptazine, ethylmethylthiambutene, ethylmorphine,etonitazene, fentanyl, heroin, hydrocodone, hydromorphone,hydroxypethidine, isomethadone, ketobemidone, levallorphan, levorphanol,levophenacylmorphan, lofentanil, loperamide, meperidine (pethidine),meptazinol, metazocine, methadone, metopon, morphine, myrophine,nalbuphine, narceine, nicomorphine, norlevorphanol, normethadone,nalorphine, normorphine, norpinanone, opium, oxycodone, oxymorphone,papaveretum, pentazocine, phenadoxone, phenomorphan, phanazocine,phenoperidine, piminodine, piritramide, propheptazine, promedol,properidine, propiram, propoxyphene, sulfentanil, tilidine, tramadol,diastereoisomers thereof, pharmaceutically acceptable salts thereof,complexes thereof; and mixtures thereof. In some embodiments, the opioidis selected from morphine, codeine, oxycodone, hydrocodone,dihydrocodeine, propoxyphene, fentanyl, tramadol, and mixtures thereof.

The opioid component of the combination therapy may further include oneor more other active ingredients that may be conventionally employed inanalgesic and/or cough-cold-antitussive combination products. Suchconventional ingredients include, for example, aspirin, acetaminophen,phenylpropanolamine, phenylephrine, chlorpheniramine, caffeine, and/orguaifenesin. Typical or conventional ingredients that may be included inthe opioid component are described, for example, in the Physicians' DeskReference, 1999, the disclosure of which is hereby incorporated hereinby reference, in its entirety.

The opioid component may further include one or more compounds that maybe designed to enhance the analgesic potency of the opioid and/or toreduce analgesic tolerance development. Such compounds include, forexample, dextromethorphan or other NMDA antagonists (Mao et al., 1996,Pain 67:361), L-364,718 and other CCK antagonists (Dourish et al., 1988,Eur. J. Pharmacol 147:469), NOS inhibitors (Bhargava et al., 1996,Neuropeptides 30:2), PKC inhibitors (Bilsky et al., 1996, J. Pharmacol.Exp. Ther. 277:484), and dynorphin antagonists or antisera (Nichols etal., 1997, Pain 69:317). The disclosures of each of the foregoingdocuments are hereby incorporated herein by reference, in theirentireties.

Alternatively, beloxepin and/or its analogs may be used with at leastone non opioid analgesic, such as for example, diclofenac, a COX2inhibitor, aspirin, acetaminophen, ibuprophen, naproxen, and the like,and mixtures thereof.

Other agents that may be used in combination with the beloxepin and/orits analogs include anti-nflammatories. Specific examples of suitableanti-inflammatories include, but are not limited to, corticosteroids,aminoarylcarboxylic acid derivatives such as, but not limited to,etofenamate, meclofenamic acid, mefanamic acid, niflumic acid;arylacetic acid derivatives such as, but not limited to, acemetacin,amfenac cinmetacin, clopirac, diclofenac, fenclofenac, fenclorac,fenclozic acid, fentiazac, glucametacin, isozepac, lonazolac, metiazinicacid, oxametacine, proglumetacin, sulindac, tiaramide and tolmetin;arylbutyric acid derivatives such as, but not limited to, butibufen andfenbufen; arylcarboxylic acids such as, but not limited to, clidanac,ketorolac and tinoridine; arylpropionic acid derivatives such as, butnot limited to, bucloxic acid, carprofen, fenoprofen, flunoxaprofen,ibuprofen, ibuproxam, oxaprozin, piketoprofen, pirprofen, pranoprofen,protizinic acid and tiaprofenic add; pyrazoles such as, but not limitedto, mepirizole; pyrazolones such as, but not limited to, clofezone,feprazone, mofebutazone, oxyphenbutazone, phenylbutazone, phenylpyrazolidininones, suxibuzone and thiazolinobutazone; salicylic acidderivatives such as, but not limited to, bromosaligenin, fendosal,glycol salicylate, mesalamine, 1-naphthyl salicylate, olsalazine andsulfasalazine; thiazinecarboxamides such as, but not limited to,droxicam, isoxicam and piroxicam; and other anti-inflammatory agentssuch as, but not limited to, e-acetamidocaproic acid,s-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,bendazac, bucolome, carbazones, difenpiramide, ditazol, guaiazulene,heterocyclic aminoalkyl esters of mycophenolic acid and derivatives,nabumetone, nimesulide, orgotein, oxaceprol, oxazole derivatives,paranyline, pifoxime,2-substituted-4,6-di-tertiary-butyl-s-hydroxy-1,3-pyrimidines,proquazone and tenidap.

Beloxepin and its analogs can also be used in combination with eachother. Thus, in some embodiments, the combination therapy involvesadministration of two or more beloxepin analogs, or beloxepin and one ormore beloxepin analogs.

8.5 Additional Properties of Beloxepin

As indicated in Example 3, an initial, screening study suggested thatbeloxepin inhibits the polymorphic cytochrome P450 isoenzyme CYP2D6(IC₅₀=536 nM). A subsequent, more definitive analysis in which CYP2D6inhibition by beloxepin was measured human hepatic microsomes usingdextromethorphan as the model. There, beloxepin caused direct inhibitionof CYP2D6 with an IC₅₀ value of only 31.7 μM (FIG. 15), indicating that,CYP inhibition would therefore be negligible for beloxepin. CytochromeP450 enzymes play important roles in drug metabolism. For example, manytricyclic antidepressants used off-label to treat pain are metabolizedby CYP2D6. Use of inhibitors of this enzyme in combination therapyregimens can therefore dramatically increase their levels.Co-administration of CYP2D6 inhibitors with substrates of CYP2D6 canalso prolong the QT interval, leading to arrythmias.

Certain prodrugs are acted upon by CYP2D6 to release the active drug.CYP2D6 inhibitors would likely reduce the efficacy of suchCYP2D6-activated drugs. As a specific example, clinical evidence suggestthat CYP2D6-activated prodrugs such as codeine and tramadol are lesseffective in patients who are genetically deficient in CYP2D6 or inpatients receiving potent CYP2D6 inhibitors.

Cytochrome P4502D6 (CYP2D6) is a polymorphic member of the P450superfamily, which is absent in 5-9% of the Caucasian population,resulting in a deficiency in drug oxidation known asdebrisoquine/sparteine polymorphism. Metabolism by polymorphicisoenzymes such as CYP2D6 can be problematic in drug development becauseof the wide variation in the pharmacokinetics of the patient population.CYP2D6 metabolises many currently used drugs, which include β-blockers,antidepressants, and neuroleptics (Bertz and Granneman, 1997, Clin.Pharmokinet. 32(3):210-58). Polymorphisms of 2D6 have been associatedwith a reduced capacity to dispose important drugs; this leads toundesirable clinical consequences (Ingelman-Sundberg et al., 1999,Trends. Pharmacol. Sci. 20(8):342-349). The impact of human P450polymorphisms on drug treatment in poor metabolizers is indicated inTable 1 below (Ingelman-Sundberg et al., 1999, Trends. Pharmacol. Sci.20(8):342-349).

TABLE 1 Impact of human P450 polymorphisms on drug treatment in poormetabolizers Polymorphic Decreased Reduced prodrug enzyme clearanceAdverse effects activation CYP 2C9 S-Warfarin Bleeding LosartanPHenytoin Ataxia Losartan Tolbutamide Hypoglycaemia NSAIDs GI bleedingCYP 2C19 Omeprazole Proguanil Diazepam Sedation CP2D6 TricyclicCardiotoxicity Tramadol antidepressants Codeine Haloperidol ParkinsonismEthylmorphine Anti-arrhythmic Arrhythmias drugs Perphenazine PerhexilineNeuropathy SSRIs Nausea Zuclopenthixol S-Mianserin TolterodineAbbreviations: NSAIDs, nonsteroidal anti-inflammatory drugs; SSRIs,selective serotonin reuptake inhibitors

Thus, in view of the above and the data of Example 3, skilled artisanswill appreciate that in the various combination therapies discussedherein, dosages may need to be adjusted when beloxepin and/or itsanalogs are administered in combination with, or adjunctively to, drugsthat are either metabolized by or activated by, CYP2D6.

As indicated above, preliminary screening assays for inhibition ofcDNA-expressed human CYP450 isozymes by beloxepin at 10 μM, suggestedextensive inhibition of CYP2D6 (97%). The potential inhibition of CYP2D6was re-evaluated using dextromethorphan as the model substrate, andmeasuring inhibition of CYP2D6 by beloxepin in human hepatic microsomes.In these definitive studies, beloxepin caused direct inhibition ofCYP2D6 with an IC₅₀ value of 31.7 μM (FIG. 15). At anticipatedtherapeutic plasma concentrations, CYP inhibition would therefore benegligible for beloxepin. This suggests that beloxepin has littlepotential for drug-drug interactions.

8.6 Formulations And Administration

Beloxepin and/or its analogs (or salts thereof) may be combined with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice as described, forexample, in Remington's Pharmaceutical Sciences, 2005, the disclosure ofwhich is hereby incorporated herein by reference, in its entirety. Therelative proportions of active ingredient and carrier may be determined,for example, by the solubility and chemical nature of the compounds,chosen route of administration and standard pharmaceutical practice.

The compositions may be formulated for oral administration, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompound may be incorporated with excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The amount of activecompound(s) in such therapeutically useful compositions is preferablysuch that a suitable dosage will be obtained. Preferred compositions orpreparations may be prepared so that an oral dosage unit form containsfrom about 0.1 to about 1000 mg of each beloxepin enantiomer (and allcombinations and subcombinations of ranges and specific concentrationstherein).

The tablets, troches, pills, capsules and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient, such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, lactose or saccharin; or a flavoring agent such aspeppermint, oil of wintergreen or cherry flavoring. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, a liquid carrier. Various other materials may be present ascoating, for instance, tablets, pills, or capsules may be coated withshellac, sugar or both. A syrup or elixir may contain the activecompound, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form ispreferably pharmaceutically pure and substantially non toxic in theamounts employed.

The compositions may also be formulated for parenteral orintraperitoneal administration. Solutions of the beloxepin enantiomersas free bases or pharmacologically acceptable salts can be prepared inwater suitably mixed with a surfactant, such as hydroxypropylcellulose.A dispersion can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Compositions suitable for administration by injection typically include,for example, sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases, the form is preferably sterileand fluid to provide easy syringability. It is preferably stable underthe conditions of manufacture and storage and is preferably preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, liquid polyethylene glycol and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of a dispersion, and by theuse of surfactants. The prevention of the action of microorganisms maybe achieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions may be achieved by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in the required amounts, in the appropriate solvent, withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions may be prepared byincorporating the sterilized active ingredient into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation may include vacuum drying and the freeze dryingtechnique that yields a powder of the active ingredient, plus anyadditional desired ingredient from the previously sterile filteredsolution thereof.

8.7 Effective Dosages

Beloxepin and/or beloxepin analogs will generally be administered in atherapeutically effective amount, as described herein. The quantity ofbeloxepin and/or beloxepin analog compounds will depend upon a varietyof factors, including, for example, the particular pain indication orsyndrome being treated, the mode of administration, whether the desiredbenefit is prophylactic or therapeutic, the severity of the painindication or syndrome being treated, the age and weight of the patient,and the bioavailability of beloxepin and/or beloxepin analog(s)administered. Determination of an effective dosage is well within thecapabilities of those skilled in the art.

Dosage amounts will typically be in the range of from about 0.0001 or0.001 or 0.01 mg/kg/day total active compound(s) to about 0.1 or 1.0 or2.0 or 2.5 or 5.0 or 10.0 or 20.0 or 25.0 or 50.0 or 75.0 or 100mg/kg/day total active compound(s), with an expected dose of about 5mg/kg/day to about 1500 mg/kg/day total active compound(s), but may behigher or lower, depending upon, among other factors, the factorsmentioned above.

Dosage amount and interval may be adjusted individually to provideplasma levels of active compound(s), which are sufficient to maintaintherapeutic or prophylactic effect. As non-limiting examples, thecompositions may be administered once per day or multiple times per day,depending upon, among other things, the mode of administration, thespecific indication being treated and the judgment of the prescribingphysician. In cases of local administration or selective uptake, such aslocal topical administration, the effective local concentration ofactive compounds and/or compositions may not be related to plasmaconcentration. Skilled artisans will be able to optimize effective localdosages without undue experimentation.

Based on the animal data described in the Examples section, it isexpected that an effective dosage of beloxepin for the treatment of painin humans may be obtained by administering a dose of beloxepinsufficient to achieve a plasma concentration similar to that achievedfollowing the administration of 30 mg/kg, i.p. to rats, or 60 mg/kg POto rats. As such, in some embodiments the effective dose of beloxepinfor the treatment of pain is the dosage required to achieve the plasmaconcentration achieved when 30 mg/kg beloxepin is administered i.p. torats, or when 60 mg/kg beloxepin is administered orally to rats.

Based on these animal data, it is expected that oral doses of beloxepinof between about 10 mg/day to about 20 or 25 or 30 or 35 or 40 or 45 or50 or 60 or 70 or 80 or 90 or 95 or 100 or 200 or 500 or 750 or 1000 or1500 mg/day will be effective in treating pain. Accordingly, someembodiments involve the administration of an oral dosage of beloxepinthat ranges from about 10 mg/day to about 500 mg per dose, one or moretimes per day. It is expected that similar dosage ranges of beloxepinanalogs will be effective.

In the context of combination therapy, the proper dosage of the combinedagents will be readily ascertainable by a skilled artisan based on longestablished criteria. By way of general guidance, where a cannabinoid,opioid and/or other agent is used in combination with beloxepin, thedosage will typically range from about 0.01 to about 100 mg/kg/day ofthe cannabinoid, opioid and/or other active compound and about 0.001 toabout 100 mg/kg/day of beloxepin. In certain embodiments, the dosage maybe about 0.1 to about 10 mg/kg/day of the cannabinoid, opioid and/orother active compound and about 0.01 to about 10 mg/kg/day of beloxepin,and in other embodiments, the daily dosage may be about 1.0 mg of thecannabinoid, opioid and/or other active compound and about 0.1 mg ofbeloxepin. Alternatively, when beloxepin is combined with a cannabinoidcompound (e.g., Δ⁹-tetrahydrocannabinol or cannabidiol), an opioidcompound (e.g., morphine) and/or an other agent and the combination isadministered orally, the dosage may generally range from about 15 toabout 200 mg of the cannabinoid, opioid and/or other agent, and about0.1 to about 4 mg of beloxepin. It is expected that similar dosageranges will be effective for combination therapies with beloxepinanalogs.

8.8 Kits

Beloxepin and/or beloxepin analogs may be assembled in the form of kits.In some embodiments, the kit provides the compounds(s) and reagents toprepare a composition for administration. The composition may be in adry or lyophilized from, or in a solution, particularly a sterilesolution. When the composition is in a dry form, the reagent maycomprise a pharmaceutically acceptable diluent for preparing a liquidformulation. The kit may contain a device for administration or fordispensing the compositions, including, but not limited to, syringe,pipette, transdermal patch or inhalant.

The kits may include other therapeutic agents for use in conjunctionwith the compositions described herein. In some embodiments, thetherapeutic agents may be provided in a separate form, or mixed with thecompositions described herein.

Kits can include appropriate instructions for preparation andadministration of the composition, side effects of the compositions, andany other relevant information. The instructions may be in any suitableformat; including, but not limited to, printed matter, videotape,computer readable disk, or optical disk.

9. EXAMPLES

The following working examples, which are intended to be illustrativeand not limiting, highlight various features of beloxepin and certainuses described herein.

Example 1 Synthesis of (±)-Beloxepin

With reference to Scheme 1, reproduced below, beloxepin was synthesizedas follows.

Preparation of 2-(2-(o-tolyloxy)phenyl)acetic acid (B): To a solution ofA (50.0 g, 232 mmol, 1.00 eq) in N,N-dimethylformamide (500 mL) undernitrogen and with mechanical stirring was added cesium carbonate (189 g,581 mmol, 2.50 eq), o-cresol (28.8 mL, 279 mmol, 1.20 eq), copper(I)chloride (12 g, 120 mmol, 0.5 eq) and tris(3,6-dioxaheptyl)amine (TDA)(37 mL, 120 mmol, 0.5 eq). The reaction was degassed by bubblingnitrogen through the stirring mixture for 10 minutes. The mixture wasthen heated at 80° C. for 2 days under nitrogen. The reaction was cooledto room temperature and diluted with 1:1 diethyl ether/hexanes. Whilestirring, the mixture was carefully acidified with 6M HCl, then dilutedwith water and the layers were separated. The aqueous layer was washedwith 1:1 diethyl ether/hexanes and all organics were combined and washedwith 0.5M sodium carbonate. The basic aqueous layers were combined,acidified with 6M HCl and the product was extracted with diethyl ether.The organics were concentrated and purified by a silica gel plug using2-5% isopropanol/hexane gradient to give 31.48 g yellow/green oil (51%yield, based on ¹H NMR purity of 92%). ¹H NMR (400 MHz, CDCl₃) 7.29 (dd,1H), 7.23-7.10 (m, 3H), 7.05 (m, 2H), 6.83 (dd, 1H), 6.63 (dd, 1H), 3.77(s, 2H), 2.20 (s, 3H); MS: (M−H)⁻=241.1.

Preparation of 6-methyldibenzo[b,f]oxepin-10(11H)-one (C): A mixture ofB (60.7 g, 213 mmol, 1.00 eq, 85% purity), polyphosphoric acid (93 g,852 mmol, 4.00 eq) and sulfolane (200 mL) was immersed in an oil bath at120° C. and heated for 90 minutes. Ice water was added and the productwas extracted with diethyl ether. The organic layer was washed with 0.5M sodium carbonate, concentrated and purified by a silica gel plug usinga 1-4% ethyl acetate/hexanes gradient to give 41.4 g orange oil (80%**).**Yield based on 85% purity of starting material B and 92% purity ofproduct C. ¹H NMR (400 MHz, CDCl₃) 7.91 (m, 1H), 7.44 (m, 1H), 7.32 (m,1H), 7.25 (m, 2H), 7.19 (m, 1H), 7.07 (m, 1H), 4.10 (s, 2H), 2.57 (s,3H)

Preparation of(4-Methyl-11-oxo-10,11-dihydro-dibenzo[b,f]oxepin-10-yl)-acetic acidtert-butyl ester (D): To a mixture of 60% sodium hydride in mineral oil(8.16 g, 204 mmol, 1.2 eq) in tetrahydrofuran (400 mL) cooled in abrine/water bath was added dropwise a solution of the ketone C (41.4 g,170 mmol, 1.0 eq, 92% purity) in tetrahydrofuran (200 mL). The mixturewas stirred for an additional 10 minutes. The bromide was added dropwiseover a 10 minutes period and the reaction was stirred cooled for 40minutes. The reaction was quenched with water and concentrated. Thecrude product was partitioned between water and diethyl ether, layerswere separated and the organics were washed with brine. The organicswere concentrated and the resulting solid was triturated in hexanes,filtered and dried to give 44.1 g of an off-white solid. The filtratewas concentrated and there were crystals after 3 days. Crystals werefiltered and dried to give 1.5 g pale orange crystalline solid. Totalyield=78%. ¹H NMR (400 MHz, CDCl₃) 7.86 (dd, 1H), 7.43 (m, 1H),7.25-7.20 (m, 4H), 7.06 (t, 1H), 4.83 (m, 1H), 3.37 (m, 1H), 2.87 (dd,1H), 2.57 (s, 3H), 1.42 (s, 9H); MS: M⁺=338.4

Preparation of(4-Methyl-11-oxo-10,11-dihydro-dibenzo[b,f]oxepin-10-yl)-acetic acid(E): The ester D (44.0 g, 128 mmol, 1.0 eq) was dissolved indichloromethane (500 mL) and trifluoroacetic acid (34.5 mL, 448 mmol,3.5 eq) was added. The reaction was stirred at room temperature over 48h. The reaction was diluted with water and the layers were separated.The organics were concentrated, triturated in 1:1 diethyl ether/hexanes(250 mL), filtered and dried to give 34.6 g of a pale yellow solid(94%). ¹H NMR (400 MHz, DMSO) 12.40 (brs, 1H), 7.72 (dd, 1H), 7.61 (m,1H), 7.44 (m, 1H), 7.36-7.30 (m, 3H), 7.18 (t, 1H), 4.73 (m, 1H), 3.33(m, 1H), 2.92 (dd, 1H), 2.57 (s, 3H); MS: (M−H)⁻=281.2

Preparation ofN-Methyl-2-(4-methyl-11-oxo-10,11-dihydro-dibenzo[b,f]oxepin-10-yl)-acetamide(F): The acid E (34.5 g, 120 mmol, 1.0 eq) was suspended intetrahydrofuran (200 mL) under nitrogen. To the mixture was addedN,N-diisopropylethylamine (31.3 mL, 180 mmol, 1.5 eq), methyl amine (120mL, 240 mmol, 2.0 eq) and TBTU (46.2 g, 144 mmol, 1.2 eq). The reactionwas stirred at room temperature for 2 hours. Between 30 and 60 minutes,a thick precipitate forms and the reaction turns light green. Another100 mL of tetrahydrofuran was added and slow stirring resumed.N,N-dimethylformamide (100 mL) was added followed by additional amountof TBTU (15 g). The reaction mixture was concentrated to near drynessand the product was partitioned between diethyl ether and a 50% aqueoussolution of sodium bicarbonate. The aqueous was washed with diethylether and all organics were combined and concentrated. The resultingsolid was triturated in 300 mL 1:1 diethyl ether/hexanes, filtered anddried to give 33.3 g off-white solid (93%). ¹H NMR (400 MHz, CDCl₃) 7.84(dd, 1H), 7.43 (m, 1H), 7.25-7.20 (m, 3H), 7.16 (m, 1H), 7.06 (t, 1H),4.96 (dd, 1H), 3.33 (m, 1H), 2.82 (d, 3H), 2.75 (dd, 1H), 2.57 (s, 3H);MS: (M+H)⁺=296.0

Preparation of2-(11-Hydroxy-4-methyl-10,11-dihydro-dibenzo[b,f]oxepin-10-yl)-N-methyl-acetamide(G): The ketone F (33.2 g, 112 mmol, 1.0 eq) was partially dissolved inmethanol/tetrahydrofuran (200 mL/200 mL) under nitrogen and cooled in anice/water bath. Sodium borohydride (10.6 g, 281 mmol, 2.5 eq) was addedin 2 g portions over a 15 minutes period. The ice bath was removed andthe mixture was stirred at room temperature for 1 hour. The reaction wasquenched with water and concentrated to near dryness. The crude productwas suspended in dichloromethane, water was added and the layers wereseparated. The aqueous layer was washed again with dichloromethane andthe organics were combined and concentrated. To the resulting foam wasadded 250 mL of 1:1 diethyl ether/hexanes with vigorous stirring. Awhite precipitate immediately formed and it was filtered and dried togive 32 g of a white powder (97%); MS: (M+H)⁺=298.0

Preparation of6-Methyl-11-(2-methylamino-ethyl)-10,11-dihydro-dibenzo[b,f]oxepin-10-ol(H): The amide G (31.9 g, 107 mmol, 1.0 eq) was dissolved intetrahydrofuran (200 mL) under nitrogen and the borane-dimethyl sulfidecomplex (2.0 M in tetrahydrofuran, 161 mL, 322 mmol, 3.0 eq) was addeddropwise over 15 minutes. The reaction was then heated at 80° C. for 24hours. The reaction was cooled in an ice/water bath and methanol (50 mL)was added in 10 mL portions over 30 minutes. The mixture was stirred for30 minutes at room temperature. A solution of 4M HCl in dioxane (130 mL,˜5 eq) was added dropwise over 15 minutes. The mixture was stirred atroom temperature for 30 minutes. The mixture was concentrated to neardryness and water and 10% ethyl acetate/diethyl ether were added. Layerswere separated and the aqueous phase was washed with 10% ethylacetate/diethyl ether. The aqueous layer was basified with a saturatedsodium bicarbonate solution and the product was extracted with 10%methanol/dichloromethane. The organics were combined, dried over sodiumsulfate, concentrated and dried to give 25.8 g of a yellow oil (82%).MS: (M+H)⁺=284.0

Preparation of[2-(11-hydroxy-4-methyl-10,11-dihydro-dibenzo[b,f]oxepin-10-yl)-ethy]-methyl-carbamicacid tert-butyl ester (I): To a solution of the amine H (25.0 g, 86mmol, 1.0 eq, 96.9% pure) and triethylamine (14.3 mL, 102 mmol, 1.2 eq)in dichloromethane (300 mL) was added di-tert-butyldicarbonate (19.6 g,90 mmol, 1.05 eq) portion wise. The reaction was stirred at roomtemperature for 15 minutes. The reaction was diluted with 0.5 M HCl andthe layers were separated. The organics were washed with 0.5 M HCl,dried over sodium sulfate, concentrated and dried to give 35 g of ayellow oil (100% yield based on 93% purity).

MS: (M+H)⁺=384.0

Preparation ofmethyl-[2-(4-methyl-dibenzo[b,f]oxepin-10-yl)-ethyl]-carbamic acidtert-butyl ester (J): The alcohol I (23.5 g, 57 mmol, 1.0 eq, 93%purity) was dissolved in dichloromethane (300 mL) and triethylamine(20.6 mL, 148 mmol, 2.6 eq) was added. The mixture was cooled in an icebath and methanesulfonyl chloride (5.73 mL, 74 mmol, 1.3 eq) was added.The reaction mixture was stirred cooled for 15 minutes. The reactionmixture was diluted with 0.5 M HCl and the layers were separated. Theorganics were concentrated and dried to give 28 g of a crude lightyellow oil. The mesylate was dissolved in toluene (200 mL) and1,8-diazabicyclo[5.4.0]undec-7-ene (42.6 mL, 285 mmol, 5.0 eq) wasadded. The mixture was heated at 115° C. for 1 hour and diluted withwater. The layers were separated and the organics were concentrated andpurified by a silica gel plug eluting with 5-15% ethyl acetate/hexanesto give 14.76 g of a light yellow oil. This total amount was collectedin two batches (8.44 g, 81% pure by LC/MS) and (6.32 g, 77% pure byLC/MS). ¹H NMR (400 MHz, CDCl₃) 7.40 (brm, 1H), 7.28 (m, 1H), 7.22-7.10(m, 3H), 6.98 (m, 2H), 6.70 (brs, 1H), 3.39 (brm, 2H), 2.91-2.82 (brm,5H), 2.53 (s, 3H), 1.46 (s, 9H); MS: (M+H)⁺=366.0

Preparation ofmethyl-[2-(4-methyl-dibenzo[b,f]oxepin-10-yl)-ethyl]-amine (K): Theolefin J (14.8 g, 32 mmol, 1.0 eq, 79% pure) was dissolved indichloromethane (150 mL) and a solution of HCl in diethyl ether (2.0M,75 mL, 160 mmol, 5 eq) was added. The mixture was stirred overnight atroom temperature. The reaction was diluted with a solution of saturatedsodium bicarbonate and layers were separated. The aqueous layer waswashed with 10% methanol/dichloromethane and all organics were combined,concentrated and purified by a flash silica gel column using a 2-10%methanol/dichloromethane gradient (plus 1% NH₄OH) to give 8.0 g of ayellow oil in 91% yield and 96% purity. ¹H NMR (400 MHz, CDCl₃) 7.38 (m,1H), 7.30 (m, 2H), 7.15 (m, 2H), 6.99 (m, 2H), 6.74 (s, 1H), 2.93 (t,2H), 2.78 (t, 2H), 2.52 (s, 3H), 2.44 (s, 3H); MS: (M+H)+=266.0

Preparation of Beloxepin (L): To the amine K (7.0 g, 25 mmol, 1.0 eq)under nitrogen was added ethanol (23 mL), an aqueous solution of HCl(2.0 M, 226 mL, 19 eq) and an aqueous solution of formaldehyde (37%, 100mL, 52 eq). The reaction mixture was heated at 50° C. for 64 hours. Thereaction mixture was cooled in an ice bath and it was basified with 2MNaOH to pH ˜8. The product was extracted with 10%methanol/dichloromethane. The organics were combined, concentrated andpurified by a flash silica gel column using a 4-9%methanol/dichloromethane gradient (plus 1% NH₄OH) to give 4.9 g whitesolid in 66% yield and 100% purity. ¹H NMR (400 MHz, CDCl₃) 7.62 (d,1H), 7.27 (m, 3H), 7.14 (m, 1H), 7.08 (m, 1H), 7.00 (m, 1H), 3.28 (brs,1H), 3.10 (brt, 1H), 3.00 (brm, 1H), 2.82 (brm, 1H), 2.46 (brs, 1H),2.42 (s, 3H), 2.29 (s, 3H), 2.18 (m, 1H), 2.03 (s, 1H), 1.80 (brm, 1H);MS: (M+H)⁺=296.0. CHN Theory (1 mol H₂O): % C, 72.82; % H, 7.40; % N,4.47. CHN Actual (1 mol H₂O): % C, 72.69; % H, 7.29; % N, 4.48.

Preparation of Reconstituted Racemic Mixture of Beloxepin (See FIG. 9):

300 mg of (+)-Beloxepin and 300 mg of (−)-Beloxepin were combined anddissolved in 10 mL of hexanes/methanol (30:70). The solution wasconcentrated on a rotovap at 37° C. to give an off-white foam (Beloxepinlot 9). ¹H NMR (400 MHz, CDCl₃) consistent for product. LC/MS:ESI+M+=295.6; purity=100% RT=0.64; CHN Theory: % C, 77.26; % H, 7.17; %N, 4.74. CHN Found: % C, 77.04; 77.10; % H, 7.17; 7.20; % N, 4.77; 4.79.

Example 2 Beloxepin is an Inhibitor of NE Reuptake

The binding affinities of beloxepin for the NE, serotonin and dopaminetransporters were determined in competitive binding assays withradiolabeled ligands. The ability of beloxepin to inhibit reuptake of NEwas also determined. It was observed that beloxepin had only marginalaffinity for the serotonin transporter (27% inhibition of binding at 10μM in a competition assay) and dopamine transporter (16% inhibition ofbinding at 10 μM in a competition assay). Other results observed areprovided below.

Protocols. For the NE transporter binding assay, [³H]nisoxetine (1.0 nM)was incubated with various concentrations of beloxepin for 2 hours at 4°C. with membranes prepared from Chinese hamster ovary cells (CHO) cellsheterologously expressing the cloned human NE transporter (hNET). Boundradioactivity was determined by scintillation spectroscopy. Non-specificbinding was defined as the amount of binding that occurred in thepresence of 1.0 μM desipramine. The K_(i) was determined using standardmethods.

The IC₅₀ of NE reuptake inhibition was determined by measuring thedegree to which various concentrations of beloxepin inhibitedincorporation of [³H]norepinephrine into rat hypothalamus synaptosomes(measurements carried out for 20 minutes at 37° C.).

For the 5HT transporter binding assay, [³H] imipramine (2.0 nM) wasincubated in the presence of various concentrations of beloxepin for 1hour at 22° C. with membranes prepared from CHO cells heterologouslyexpressing the human serotonin transporter (hSERT). Bound radioactivitywas determined by scintillation spectroscopy. Non-specific binding wasdefined as the amount of binding that occurred in the presence of 10 μMimipramine. The K_(i) was determined using standard methods.

The IC₅₀ of 5HT reuptake inhibition was determined by measuring thedegree to which various concentrations of beloxepin inhibitiedincorporation of [³H]-5HT into rat brain synaptosomes (measurementscarried out for 15 min at 37° C.

For the DA transporter binding assay, [³H]N-[1-(2-benzo[b]thiophenyl)cyclohexyl]-piperidine ([³H]BTCP) (4.0 nM)was incubated in the presence of various concentrations of beloxepin for2 hr at 4° C. with membranes prepared from Chinese hamster ovary (CHO)cells heterologously expressing the cloned human dopamine transporter(hDAT). Bound radioactivity was determined by scintillationspectroscopy. Non-specific binding was defined as binding that occurredin the presence of 10 μM BTCP. The K_(i) was determined using standardmethods

The IC₅₀ of DA reuptake inhibition was determined by measuring thedegree to which various concentration of beloxepin inhibitedincorporation of [³H]-DA into rat striatum synaptosomes (measurementscarried out for 15 min at 37° C.).

Results. The K_(i)s and IC₅₀s of beloxepin for the NE, 5HT and DAtransporters are provided below, showing that beloxepin is a weak,albeit selective, inhibitor of NE reuptake.

-   -   K_(i) ^(NET)=700 nM    -   IC₅₀ ^(NE)=130 nM    -   K_(i) ^(SERT)=27% inhibition of binding at 10 μM in a        competition assay    -   K_(i) ^(DAT)=16% inhibition of binding at 10 μM in a competition        assay

Example 3 Beloxepin Inhibition of Cytochrome P450 Isoenzyme CYP2D6

Protocol. The inhibitory activity of beloxepin on cytochrome P450function was tested using the methods of Chauret (Chauret et al., 2001,Drug Metabolism and Disposition, 29(9), 1196-1200) using7-methoxy-4-(aminomethyl)-coumarin (MAMC) (Venhorst et al., 2000,European Journal of Pharmaceutical Sciences 12(2): 151-158) assubstrate. The source of the enzyme was microsomes containing humanrecombinant CYP2D6 obtained from BD Bioscience. Conversion of MAMC to7-hydroxy-4-(aminomethyl)coumarin was measured using a PerkinElmerFusion with a 390 nm excitation filter and a 460 nm emission filter.

Results. Beloxepin was found to inhibit CYP2D6 activity with an IC₅₀=536nM.

Evaluation of beloxepin as a Direct Inhibitor of Human CYP2D6(dextromethorphan O-demethylation): Microsomal Incubations for IC₅₀Estimation

Protocol: The ability of Beloxepin to inhibit dextromethorphanO-demethylation (CYP2D6) was investigated using pooled male humanhepatic microsomes. Beloxepin was incubated with human liver microsomesat concentrations of 0, 0.1, 0.3, 1, 3, 10, 30 and 100 μM Beloxepin. The200 μL incubations were conducted in duplicate in 0.1 M potassiumphosphate buffer (pH 7.4) with 0.02 mg of microsomal protein, 3 mMMgCl₂, 1 mM EDTA and 7.5 μM of the probe substrate dextromethorphan in a96-well polypropylene plate maintained at 37° C. After a 3-minutepre-incubation, the reaction was initiated with the addition of 2 mMNADPH. Upon completion of the 10-minute incubation period, aliquots of100 μL were removed and added to a new plate containing 100 μL ofinternal standard in acidified acetonitrile to stop the reaction. Thequenched samples were vortexed and the precipitated protein was removedby centrifugation. Supernatant aliquots of 100 μL were transferred to LCvials and 5 μL were injected onto the HPLC system for LC/MS/MS analysisof the metabolite dextrorphan. Standards and quality control sampleswere similarly prepared using authentic dextrorphan standards.

Analytical Method

Dexthorphan concentrations were determined by high performance liquidchromatography with tandem mass spectrometric detection (LC/MS/MS) afterprotein precipitation with acidified acetonitrile containing internalstandard. Separations were performed with a Flux Rheos 2000 quaternarypump (Leap Technologies, Inc., Carrboro, N.C.) using an XTerra® MS C₁₈,3.5 μm, 4.6×50 mm column (Waters Corporation, Milford, Mass.).Dextrorphan and the internal standard were eluted with 10 mM ammoniumformate with 0.1% formic acid: 0.1% formic acid in acetonitrile (80:20,v/v) run under gradient conditions at 1.0 mL/min. A MDS Sciex API4000(Applied Biosystems, Foster City, Calif.) triple quadrupole massspectrometer equipped with a Turbo Ionspray ionization source was usedas the detector. The instrument was operated in positive ion mode usingmultiple reaction monitoring (MRM) with specific precursor-product ionpairs for dextrorphan and the internal standard. The mass transitionswere m/z 280.2>262.2 for the internal standard and m/z 258.2>157.0 fordextrorphan. Dextrorphan and the internal standard had retention timesof approximately 1.54 and 2.00 minutes, respectively.

Results. In this assay (dextromethorphan O-demethylation), Beloxepin wasfound to inhibit CYP2D6 activity with an IC₅₀=31.7 μM (FIG. 15).

Example 4 Beloxepin is Effective in Treating Neuropathic Pain

Preparation of Vehicle and beloxepin formulations. For this Example andall that follow, unless indicated otherwise, beloxepin formulations forinjection were prepared using acidified sterile water for injection(SWIJ) as a diluent. To start, a few drops (never more than 400 μl for afinal volume of approximately 14 ml) of 1 M HCl was added to neatbeloxepin. Glass beads were added and the solution vortexed vigorouslyfor 2-3 minutes, followed by sonication in a water bath for 3-5 minutesto break up larger particles. The SWIJ was then added to QS to finaltotal volume, the formulation vortexed for 2-3 minutes and thensonicated in warm water for approximately 30-60 minutes. Beloxepin wasformulated as a 10 mg/ml solution.

For this Example and all that follow, unless indicated otherwise,control vehicle was prepared using the same volumes of 1 M HCl and SWIJdiluent as the test beloxepin formulation.

Protocol. The antiallodynic activity of beloxepin was tested in vivousing the L5-Single Nerve Ligation (“SNL”) model of non-nociceptiveneuropathic pain as described in LaBuda & Little, 2005, J. Neurosci.Methods 144:175-181. The test animals were placed in a Plexiglas chamber(10 cm×20 cm×25 cm) and habituated for 15 minutes. The chamber waspositioned on top of a mesh screen so that von Frey monofilaments couldbe presented to the plantar surface of both hindpaws. Measurement oftactile sensitivity for each hind paw were obtained using the up/downmethod (Dixon, 1980, Annu. Rev. Pharmacol. Toxicol. 20:441-462) withseven Frey monofilaments (0.4, 1, 2, 4, 6, 8 and 15 grams). Each trialstarted with a von Frey force of 2 grams delivered to the right hind pawfor approximately 1-2 seconds and then the left hind paw. If there wasno withdrawal response, the next higher force was delivered. If therewas a response, the next lower force was delivered. This procedure wasperformed until no response was made at the highest force (15 grams) oruntil four stimuli were administered following the initial response. The50% paw withdrawal threshold for each paw was calculated using thefollowing formula: [Xth] log=[vFr] log+ky, where [vFr] is the force ofthe last von Frey used, k=0.2249 which is the average interval (in logunits) between the von Frey monofilaments, and y is a value that dependsupon the pattern of withdrawal responses (Dixon, 1980, supra). If ananimal did not respond to the highest von Frey monofilament (15 grams),then the paw was assigned a value of 18.23 grams. Testing for tactilesensitivity was performed twice and the mean 50% withdrawal valueassigned as the tactile sensitivity for the right and left paws for eachanimal. All test groups contained at least six animals.

Results. The antiallodynic effects produced by beloxepin (30 mg/kg IP)in L5 SNL rats 14 days post surgery are illustrated in FIG. 1. In thisexperiment, at 14 days post surgery, rats were treated with vehicle orbeloxepin (30 mg/kg IP) and tested for tactile allodynia at 30, 60, 120and 240 min post treatment. Vehicle-treated rats were tested at 30 minpost treatment. As illustrated in FIG. 1, beloxepin produced significantantiallodynia effects at the 30, 60 and 120 min time points, with amaximal effect at 30 min post treatment (829% of the threshold ofvehicle-treated rats). The magnitude of tactile allodynia observed atthe 30 min time point was amongst the highest the inventors haveobserved in this model. No side effects were observed followingtreatment.

Example 5 Beloxepin Exerts its Antiallodynic Effect in a Dose-DependentFashion

Protocol. A dose response experiment was performed in L5 SNL rats at 16days post surgery (3, 10 and 30 mg/kg IP beloxepin). In the experiment,animals were tested for tactile allodynia at 30 min post treatment. Thesham-operated control group, which were operated on but not subject tonerve ligation, contained 4 animals. The treatment group contained atleast six animals.

The results of the dose-response experiment are illustrated in FIG. 2.The 30 mg/kg dose produced a robust antiallodynic effect (852% of thethreshold for vehicle-treated rats, and almost equal to that of thesham-operated animals). The results observed replicated the significantantiallodynic effect observed in the time-course experiments of Example4.

Example 6 Beloxepin is Superior to NE Reuptake Inhibitors, MixedSerotonin/NE Reuptake Inhibitors and Tricyclic Antidepressants inTreatment of Neuropathic Pain

The results of a direct comparison of beloxepin with reboxetine, areillustrated in FIG. 3, and demonstrate that beloxepin is approximately4-fold more effective. Similarly, FIG. 5 depicts the results of a directcomparison of the antiallodynic effects produced by beloxepin,duloxetine, amitriptyline, and beloxepin in the rat L5 Spinal NerveLigation Model (30 mg/kg IP; * p<0.05 compared to vehicle-treated L5 SNLrats; rats were tested at 30 minutes or, for amitriptyline, 60 minutespost-drug administration). The data indicate that beloxepin was the mosteffective of the compounds tested.

Example 7 Beloxepin Therapy is Effective when Administered Orally

Protocol. A time course experiment was performed with beloxepin (60mg/kg PO) in L5 SNL rats at 8-days post surgery. Rats were tested at 30,60, 120 and 240 min post beloxepin. All test groups contained at leastsix animals.

Results. The results are provided in FIG. 4. Oral beloxepin producedsignificant and robust antiallodynic effects at the 30 and 60 min timepoints.

Example 8 Beloxepin is Effective at Treating Acute Nociceptive Pain

Protocol. The ability of beloxepin to treat acute nociceptive pain wasdemonstrated in the rat hot plate model utilizing Male Sprague-Dawleyrats (150-250 g). For the experiment, rats were acclimated to a 50° C.hot plate apparatus by gently placing them on the hot plate with allfour paws on the surface. A timer was started and the latency (inseconds) until the rat licked any of its paws was measured. A 60 secondcut-off to elicit a response was set to prevent tissue damage to thepaws. After the rats elicited the paw lick response, they were removedfrom the apparatus and returned to their home cages for at least 30minutes. Baseline paw lick latencies were determined prior to drugtreatments in an identical manner to the acclimation test. Followingdrug treatments, the rats were placed on the hot plate apparatus at theappropriate time and treatment paw lick latencies were determined Alltest groups contained at least six animals.

Results. The results of the experiment are illustrated in FIGS. 6A and6B. FIG. 6A shows the latency (in seconds) between placement on the hotplate and paw lick response. 30 and 60 mg/kg beloxepin (IP) exhibited astatistically significant robust anti-nociceptive effects, with bothdosages producing anti-nociceptive activity nearly as effective as 3mg/kg morphine. FIG. 6B shows the percentage of maximal effect achieved(% MPE) in the same experiment. The paw lick latency was used todetermine % MPE for each rat based on the following formula:

${\% \mspace{14mu} M\; P\; E} = {\left\lbrack \frac{{{Treatment}\mspace{14mu} {Latency}\mspace{14mu} \left( \sec \right)} - {{Baseline}\mspace{14mu} {Latency}\mspace{14mu} \left( \sec \right)}}{{60\mspace{14mu} \sec} - {{Baseline}\mspace{14mu} {Latency}\mspace{14mu} \left( \sec \right)}} \right\rbrack \times 100}$

Thus, any rats that reach the cut-off have obtained 100% MPE.

Example 9 Beloxepin is Effective at Treating Inflammatory Pain

Protocol. The ability of beloxepin to treat inflammatory pain was testedusing Freund's Complete Adjuvant (FCA)-induced mechanical hyperalgesiain rats. For the assay, the methods of DeHaven-Hudkins et al., 1999, J.Pharmacol. Exp. Ther. 289:494-502 were used to determine mechanicalhyperalgesia in rats 24 hours after intraplantar administration of 150μL Freund's Complete Adjuvant (FCA). To determine paw pressurethresholds, the rats were lightly restrained in a gauze wrap andpressure was applied to the dorsal surface of the inflamed anduninflamed paw with a conical piston using a pressure analgesiaapparatus (Stoelting Instruments, Wood Dale, Ill.). The paw pressurethreshold was defined as the amount of force (in grams) required toelicit an escape response using a cutoff value of 250 grams. Pawpressure thresholds were determined before and at specified times afterdrug treatment. All test groups contained at least six animals.

Results. The results are illustrated in FIG. 7. 30 mg/kg beloxepinnearly completely reversed hyperalgesia induced by the FCA.

Example 10 Beloxepin is Effective at Treating Visceral Pain

Protocol. The ability of beloxepin to treat visceral pain wasdemonstrated in a rodent model of acetic acid-induced writhing For theassay, male ICR mice (20-25 g) were treated with vehicle or testcompound orally 25 min prior to the intraperitoneal administration of0.6% of acetic acid. Five minutes after treatment with acetic acid, thenumber of writhes was counted for 10 min. A writhe is defined as theextension of both front and hind limbs with a concave stretch of theabdomen. The mean number of writhes was determined for each treatmentgroup and the percent inhibition of the vehicle response was calculatedusing the following formula:

$1 - {\left\lbrack \frac{{Number}\mspace{14mu} {of}\mspace{14mu} {writhes}\mspace{14mu} {after}\mspace{14mu} {treatment}}{{Number}\mspace{14mu} {of}\mspace{14mu} {writhes}\mspace{14mu} {in}\mspace{14mu} {vehicle}\mspace{14mu} {treated}\mspace{14mu} {mice}} \right\rbrack \times 100}$

All test groups contained at least six animals.

Results. The results are illustrated in FIG. 8. Beloxepin inhibitedacetidc acid-induced writhing in a dose-dependent fashion, with an ED₅₀of 13.3 mg/kg (oral).

Example 11 A Mixture of (+)-Beloxepin and (−)-Beloxepin is Effective inan Animal Model of Inflammatory Pain (FCA-Induced MechanicalHyperalgesia)

Protocol. A sample of (±)-beloxepin was prepared by milling the isolated(+)-beloxepin and (−)-beloxepin enantiomers together, bringing them upin solvent, and then removing the solvent (“Lot 9”). In this experiment,30 mg/kg of (±)-beloxepin (“Lot 7”) or 30 mg/kg of the reconstitutedracemic mixture (Lot 9) was administered in rats treated with FCA for 24hours. Thirty minutes after treatment with vehicle, (±)-beloxepin, orreconstituted racemic mixture, paw pressure thresholds were determined.Thirty minutes is the time of peak mechanical antihyperalgesia of(±)-beloxepin.

Results. As illustrated in FIG. 9, similar mechanical antihyperalgesic(96±16% vs. 77±11%) efficacy was observed in rats treated with(±)-beloxepin or the reconstituted racemic mixture. Thus, a chemicalentity that produces significant mechanical antihyperalgesia can beprovided as the mixture of its two component enantiomers.

Example 12 Beloxepin is Effective in an Animal Model of Neuropathic Pain(Rat L5 SNL Model)

Protocol. A time course experiment was performed with beloxepin (60mg/kg PO in L5 SNL rats at 7 days post-surgery. Rats were tested at 30,60, 120, and 240 minutes post-drug.

Results. Beloxepin produced significant antiallodynic effects at allfour time points, as illustrated in FIG. 10.

Protocol. In a further experiment with this animal model of pain, acomparison of the time courses for mechanical antiallodynia in the ratL5 SNL model for beloxepin, duloxetine (a drug approved for thetreatment of diabetic neuropathy), and esreboxetine (a compound in PhaseIII clinical trials for the treatment of fibromyalgia and diabeticneuropathy). The data obtained are depicted in FIG. 11.

Results. As demonstrated in FIG. 11, racemic beloxepin (30 mg/kg IP) wascomparable in efficacy to duloxetine (30 mg/kg IP), and the peakantiallodynic effect of racemic beloxepin was greater than that measuredin rats treated with esreboxetine (30 mg/kg IP).

Example 13 Beloxepin is Effective in an Animal Model of Post-OperativePain (Rat Hindpaw Incisional Pain Model)

Protocol. A time course experiment was performed with beloxepin in thehindpaw incision model. At 24 hours post surgery, rats received vehicleor beloxepin (30 mg/kg IP). Rats were tested for tactile allodynia at30, 60, 120 and 240 minutes after administration of beloxepin.

Results. As illustrated in FIG. 12, racemic beloxepin produced asignificant antiallodynic effect at all four time points (maximumhindpaw withdrawal threshold ˜29 grams or 544% of the threshold valuefor vehicle treated rats at the 30 minute time point). The antiallodyniceffect produced by racemic beloxepin in this assay is considered veryrobust.

Protocol. A second time course experiment was performed with racemicbeloxepin in the hindpaw incision model after oral (PO) administration.At 24 hours post-surgery, rats received vehicle or racemic beloxepin (60mg/kg PO). Rats were tested for tactile allodynia at 30, 60, 120 and 240minutes after administration of beloxepin.

Results. As illustrated in FIG. 13, racemic beloxepin produced asignificant antiallodynic effect at all four time points (maximumhindpaw withdrawal threshold ˜24 grams at the 30 and 60 minute timepoints). The antiallodynic effect produced by beloxepin in this assay isconsidered very robust and is comparable to the effect that was observedafter IP administration.

Protocol. A third time course experiment was performed with racemicbeloxepin in the hindpaw incision model after intravenous (IV)administration. At 24 hours post-surgery, rats received vehicle orbeloxepin (3 mg/kg IV). The 3 mg/kg IV dose is a dose that is 10-foldlower than a dose that produced a significant respiratory orcardiovascular side effect. Rats were tested for tactile allodynia at30, 60, 120 and 240 minutes after administration of beloxepin.

Results. As illustrated in FIG. 14, racemic beloxepin produced asignificant antiallodynic effect at the 30 and 120 minute time points(maximum hindpaw withdrawal threshold ˜21 grams at the 30 minute timepoint). The antiallodynic effect produced by beloxepin in this assay atthe 30 minute time point is considered very robust and comparable to theantiallodynic effect observed with a dose of 60 mg/kg PO of racemicbeloxepin at the 30 minute time point.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

1. A method of treating pain in a mammal, comprising administering to a mammal suffering from pain an amount of beloxepin, or a salt thereof, effective to treat the pain.
 2. The method of claim 1 in which the beloxepin is administered parenterally.
 3. The method of claim 1 in which the beloxepin is administered orally.
 4. The method of claim 1 in which the pain is acute or chronic pain of nociceptive origin.
 5. The method of claim 4 in which the pain is inflammatory pain.
 6. The method of claim 4 in which the pain is cancer pain.
 7. The method of claim 1 in which the pain is chronic pain of non-nociceptive origin.
 8. The method of claim 7 in which the pain is neuropathic pain.
 9. The method of claim 1 in which the pain is visceral pain.
 10. The method of any one of claims 1-9 in which the mammal is a human.
 11. A method of treating pain in a mammal, comprising administering to a mammal suffering from pain an amount of beloxepin and/or a beloxepin analog, or a salt thereof, effective to treat the pain.
 12. The method of claim 11 in which the pain is acute or chronic pain of nociceptive origin.
 13. The method of claim 12 in which the pain is inflammatory pain.
 14. The method of claim 12 in which the pain is cancer pain.
 15. The method of claim 11 in which the pain is chronic pain of non-nociceptive origin.
 16. The method of claim 15 in which the pain is neuropathic pain.
 17. The method of claim 11 in which the pain is visceral pain.
 18. The method of any one of claims 11-17 in which the beloxepin, beloxepin analog and/or a salt thereof, is administered to the mammal in the form of a composition.
 19. The method of claim 18 in which the beloxepin and/or beloxepin analog is included in the composition as a salt.
 20. The method of claim 18 in which the mammal is a human.
 21. The method of claim 18 in which the composition is formulated for oral administration
 22. The method of claim 21 in which the mammal is a human. 